Mitochondrial protecting agents inhibit t‐butylhydro‐peroxide‐induced and caspase‐mediated neuronal death in cultured cerebellar neurons

Abstract

AbstractMitochondrial release of cytochrome c and activation of caspases are considered to be parts of a major cell death pathway in cells of various origins. In order to study if caspases are involved in the events leading to cell death induced by oxidants we exposed cerebellar granule cells to tert‐butylhydroperoxide (t‐BuOOH). The cultured cells were treated with various concentrations of t‐BuOOH for 30 min, then washed with normal cell culture medium and allowed to recover for 3 h in the normal medium before the analyses were performed. Neuronal viability was assessed. The results showed that t‐BuOOH caused neuronal death in a dose‐dependent manner. Double‐staining confocal microscopy with antibodies against active forms of caspase‐3 or caspase‐7 and propidium iodide demonstrated that both caspase‐3 and caspase‐7 were markedly activated in cultured neurons after t‐BuOOH treatment. The activation of caspase‐3 and caspase‐7 was confirmed by Western blot analysis. The mitochondrial protecting agents cyclosporin A (CsA) and ursodeoxycholic acid (UDCA) and the free radical scavenger alpha‐phenyl‐N‐tert‐butyl nitrone (PBN) protected cerebellar granule cells against t‐BuOOH induced neuronal death and inhibited the activation of caspases. These data provide evidence that cultured cerebellar granule cells, when exposed to an oxidant such as t‐BuOOH, undergo cell death by activation of caspases. Furthermore, compounds that protect mitochondria or act as free radical scavengers may salvage the cells by blocking the death process.